A fatal case of Vibrio cholerae-associated diarrhea and bacteremia in a 30-year-old carrier of beta-thalassemia

Abstract

Bacterial infections leading to bacteremia and septicemic shock constitute an emerging public health concern globally, especially in areas where sanitation is poor and safe drinking water is scarce. Enteric pathogens such as Vibrio cholerae are responsible for many deaths caused by contaminated food and water in these areas. While cholera is the prominent clinical threat posed by V. cholerae, outcomes like bacteremia turning into sepsis and associated morbidity and mortality have been increasing globally in recent times. Here, we report an alarming case of fatal sepsis with a probable association of V. cholerae bacteremia in Bangladesh. In September 2023, a 30-year-old man with a pre-condition of beta-thalassemia presented to a tertiary care hospital with acute diarrhea, abdominal pain, nausea, and fever and died within 36 h of admission with acute cholecystitis, metabolic acidosis, acute kidney injury, pancytopenia, and refractory septic shock with multi-organ dysfunction syndrome. Blood culture detected V. cholerae, which was further characterized as hemolytic, carrying the hemolysin gene and genes for the virulence factor type-three secretion system. The isolate was confirmed as V. cholerae non-O1/O139 (NOVC), which differed in genetic properties from the few contemporary NOVC isolates associated with diarrheal cases in Bangladesh. To manage the diarrhea and septicemic condition, the patient was treated empirically with metronidazole and meropenem. However, antibiotic susceptibility testing showed the strain was susceptible to all the routinely prescribed drugs for V. cholerae infections. To the best of our knowledge, this investigation provides the first molecular description of a fatal case of V. cholerae-associated bacteremia in Bangladesh and underscores the need for comprehensive investigations on bacterial septicemia to prevent future casualties.

Introduction

Septicemia is a life-threatening infection associated with organ injury resulting from a dysregulated host response to an infection [1, 2]. Due to underreporting, especially from low- and middle-income countries (LMICs), the true magnitude of the incidents is unknown, while the actual number of deaths is often masked because fatalities are attributed to the underlying conditions instead of sepsis. A recent global analysis extrapolated the incidence and mortality rate of sepsis to 48.9 million cases and 11 million fatalities each year [1], highlighting the gravity of the issue. Of the recorded incidences, diarrheal illness appears to be the most common disease that leads to septicemia [1].

Vibrio spp. are a diverse group of organisms, including notorious human pathogens like V. cholerae, V. vulnificus, and V. parahaemolyticus, responsible for diarrheal illnesses globally that can potentially cause bacteremia leading to sepsis. The bacteria can cause illnesses, categorized into two types: cholera, caused by toxigenic V. cholerae, and non-cholera infections or vibriosis. V. cholerae is primarily known for causing cholera and gastroenteritis but occasionally can cause infections in other body sites [3]. Alarmingly, the incidences of cholera and vibriosis are on the rise globally, potentially due to global climate change and rising temperatures [4]. This rise in cases of such diseases could dramatically escalate the threat posed by the bacteria to the burden of septicemia-associated illness and death.

V. cholerae is a gram-negative bacterium ubiquitously found in aquatic ecosystems worldwide and is the cause of the pandemic disease cholera [5]. There are more than 200 different serogroups of the bacteria, of which serogroups O1 and O139 have been found to consistently conserve the ability to produce cholera toxin, hence being associated with cholera [5]. The serogroup O1 has been associated with seven pandemics of cholera, affecting millions throughout the world. As a result, strains of V. cholerae O1 and O139 have been studied extensively. Strains belonging to other serogroups, collectively designated as non-O1/O139 V. cholerae (NOVC) [6, 7], typically do not produce cholera toxin but have been found to cause small outbreaks of cholera-like diseases and sporadic intestinal and extra-intestinal illnesses [8]. However, there have been growing reports of NOVC infections in recent years, a significant number of which progressed to severe and fatal infections [4, 9,10,11,12], underscoring the threat presented by these strains. Especially when the infection progresses to bacteremia and sepsis, V. cholerae infection can result in catastrophic outcomes [3, 10, 11, 13, 14]. In Bangladesh, there is a low incidence of reported gastroenteric infections caused by NOVC, and to the best of our knowledge, there have been no reports of bacteremia caused by V. cholerae to date. Thus, the true burden remains unknown, even though observations from similar settings [15, 16] suggest that these cases are hugely underreported. Here, we report a case of V. cholerae-associated diarrhea and bacteremia in a 30-year-old man who succumbed to death within 36 h of hospitalization and present a detailed phenotypic and genotypic characterization of the isolate for future reference in such unfortunate cases.

Method and materials

The bacterium was isolated from the blood culture of the patient seeking treatment at The Evercare Hospital, Dhaka, Bangladesh. Identification of V. cholerae was performed according to standard culture and molecular methods as described previously [17]. Hemolytic property of the isolate was tested on 5% sheep blood agar plates with an overnight incubation at 37°C. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen Inc.) as per the manufacturer’s instructions. The serogroup of the V. cholerae isolate was confirmed by a slide agglutination test using V. cholerae O1 and O139-specific polyvalent antisera [17]. Molecular confirmation of serogroups, species-specific genes, and detection of virulence-associated genes namely ompW, viuB, rfbO1, rfbO139, tcpA, TTSS, hlyA, ompU, rtxC was done by Polymerase chain reaction (PCR) following methods described previously [17,18,19,20]. Using the Clinical and Laboratory Standards Institute guidelines [21], a standard disc diffusion test on Muller-Hinton agar (BD, USA) determined bacterial susceptibility to antimicrobials. Fourteen drugs of different drug classes, namely azithromycin (AZM), tetracycline (TE), ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT), chloramphenicol (C), erythromycin (E), imipenem (IPM), gentamicin (CN), aztreonam (ATM), amoxicillin-clavulanate (AMC), levofloxacin (LEV), ciprofloxacin (CIP), cefepime (FEP), ceftriaxone (CRO), meropenem (MEM), vancomycin (VA), colistin (CT), and rifampin (RD), were tested. The minimum inhibitory concentration (MIC) was determined by E-test (Biomeuriex). PCR was also performed to detect antimicrobial resistance-associated genes, namely qnrVC, mphA, sxt, bla-NDM, bla-OXA-48, using primers and procedures described elsewhere [22,23,24]. Nucleotide sequencing of viuB, gyrA, parC, and TTSS genes was performed using the Sanger di-deoxy termination method at the sequencing facility at the International Centre for Diarrheal Disease Research, Bangladesh (icddr, b). To amplify 293 bp of the viuB region from DNA extracted from water samples, a PCR reaction was performed in a total of 20 µL reaction mix using 1 µL each of 10 pmol forward and reverse primers (viuB2f 5’-CCGTTAGACAATACCGAGCAC-3’ and viuB5r 5’-TTAGGATCGCGCACTAACCAC3’), 2 µL of 10 mM dNTP mix (ThermoFisher), 1 µL of magnesium chloride (ThermoFisher), 0.2 of µL Taq DNA Polymerase (Invitrogen), 2 µL of 10× Buffer, and 2 µL of template DNA. The PCR reaction was performed using the following program: initial denaturation at 95 °C for 3 min; followed by 34 cycles of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s and extension 72 °C for 1 min; followed by a final extension at 72 °C for 5 min. Sequencing of the PCR product was performed in an Applied Biosystems 3500XL genetic analyzer using the BigDye Terminator v3.1 cycle sequencing ready reaction kit (PerkinElmer). The sequence files were processed and multiple sequence analysis was performed in Geneious (Geneious 2023.2) comparison using the existing viuB reference sequence types described in Kirchberger et al. [19].

Case history and results

A 30-year-old nondiabetic, normotensive male patient presented to the emergency department of The Evercare Hospital on 8 September 2023 with severe upper abdominal pain since morning. He also experienced nausea, several episodes of loose stool, and a fever the previous night. The patient has been diagnosed with beta-thalassemia intermedia since the age of 4 years, had gross hepatosplenomegaly, and received his last blood transfusion more than a year ago (Table 1).

Table 1 Summary of clinical laboratory tests results

At the emergency department, a physical examination of the patient revealed mild disorientation (GCS was E4 V4 M6), hypotension, tachycardia, and tachypnoea with a normal body temperature. He had generalized abdominal tenderness with reduced urinary output. His blood picture showed pancytopenia with moderate anemia (hemoglobin 7.6 g/dl), mildly elevated liver enzymes (SGOT 151 IU/L, SGPT 64 U/L), raised serum urea (52 mg/dl) and creatinine (1.85 mg/dL) and a normal electrolyte level. His septic biomarkers were suggestive of sepsis (CRP 9.80 mg/dl, procalcitonin 72.24 ng/ml), and his coagulation profile was grossly deranged. His arterial blood gas analysis revealed metabolic acidosis with a pH of 7.2. An ultrasonogram of his abdomen showed features suggestive of acute intestinal obstruction or paralytic ileus, acute cholecystitis with thick sludge, moderate abdominopelvic ascites, and bilateral trace pleural effusion. He was moved to the intensive care unit as he had acute watery diarrhea with metabolic acidosis, septic shock, acute kidney injury, and pancytopenia. There, adequate hydration was ensured under central venous pressure (CVP) guidance, and he was started on intravenous sodium bicarbonate and the antimicrobials Inj. Meropenem (1 gm thrice a day) and metronidazole (500 mg four times a day) empirically.

At 7 p.m. the same day, the patient’s condition became critical with a non-recordable pulse and blood pressure and subsequent cardiac arrest was evident. The patient was immediately taken into advanced cardiac life support with mechanical ventilation. He was also given one unit of packed red blood cell. At 11:45 p.m., his condition worsened again with non-recordable blood pressure, no pulse, and a non-detectable SPO2 with a fixed and dilated pupil. Upon medical examination, the patient was declared dead on 9 September 2023, with the multi-organ dysfunction syndrome as the main cause of death and acute cholecystitis, metabolic acidosis, acute kidney injury, beta thalassemia intermedia, pancytopenia, and refractory septic shock as secondary confounders.

Laboratory investigations

Blood culture performed at the microbiology lab of Evercare Hospital revealed the growth of V. cholerae, and the isolate was sent to the V. cholerae reference laboratory at the icddr, b for serotyping and further investigation. The isolate (SNVC-01) showed the colony morphology of a typical V. cholerae on thiosulfate citrate bile salt (TCBS) agar and taurocholate tellurite gelatine agar (TTGA) (Fig. 1). Reaction with polyvalent V. cholerae O1 and O139 antisera did not result in any agglutination of the strain; hence, it was typed as V. cholerae non-O1/O139 (NOVC).

Fig. 1

The isolate showing colony morphology of a typical V. cholerae strain demonstrating yellow colonies on TCBS agar (a) and gelatinase production on TTGA (b)

When tested for hemolytic properties, the strain had moderate beta-hemolytic activity compared to the strong hemolytic activity by the reference V. cholerae O1 El Tor strain N16961 (Fig. 2). The hemolytic properties of the strain were compared with nine other clinical NOVC strains isolated from stool sample of diarrheal patients at icddr, b laboratory. Notably, these strains exhibited a wide range of hemolytic activity, from non-hemolytic to strongly hemolytic (Table 2).

Fig. 2

The strain showing moderate hemolytic activity on 5% sheep blood agar compared to the reference strains. N16961 – Vibrio cholerae O1, ET biotype, O395 – V. cholerae O1, classical biotype, CD5 – V. cholerae non O1/O139, VC-B – SNVC 01; Non-hemolytic (-), Moderate Hemolytic Activity (+), Intermediate Hemolytic Activity (++), Strong Hemolytic Activity (+++)

Table 2 Phenotypic and genotypic characteristics of Vibrio cholerae non O1/O139 isolated from the sepsis patient and dirrheal patients in Dhaka, Bangladesh

PCR based investigation revealed that the strain carried V. cholerae species-specific genes, ompW and viuB, confirming the identity of the isolate. PCR for serogroup O1 and O139-antigen-specific genes, rfbO1 and rfbO139, gave a negative result typical of NOVC. Among the potential virulence genes tested, the isolate carried the hemolysin encoding gene hlyA, type-three secretion system (TTSS) associated genes vcsC2, vopM, and vopG, and the repeat-in toxin gene, rtxC. However, it was negative for the canonical virulence factors cholera toxin gene (ctxA), toxin co-regulated pili (tcpA), and the outer membrane porin, ompU.

To assess the subtype properties of the isolate in the context of contemporary V. cholerae isolates, the marker gene viuB was sequenced, and the genotype was determined as viuB-56 following the criteria described by Kirchberger et al. BLAST search in the NCBI nucleotide database revealed three other V. cholerae strains, including one isolated from clinical sample, to possess genotype viuB-56 (Accession no. AP028804, CP078727, and CP078723). To assess additional genotypic information, nucleotide sequencing of the PCR amplicon of the genes gyrA and parC was done. Sequence analysis revealed that the isolate SNVC-01 possessed the wild-type allele of both gyrA and parC, whereas contemporary V. cholerae isolates worldwide are commonly known to have mutations ‘Ser83 to Ile’ and ‘Ser85 to Leu’ in gyrA and parC, respectively [25, 26].

In the icddr, b laboratory, the antimicrobial susceptibility of the isolate was determined against a panel of fourteen antibiotics and compared with that of contemporary NOVC isolates. The antimicrobial susceptibility test (AST) revealed that SNVC-01 was susceptible to twelve antibiotics of different drug classes, including the drugs (doxycycline, tetracycline, erythromycin, and azithromycin) commonly prescribed for cholera (Fig. 3). The strain was resistant to colistin and vancomycin, with no visible zone of inhibition. Minimum inhibitory concentration (MIC) was measured for colistin, and the breakpoint for inhibition was 64 µg/mL. In the molecular tests, antimicrobial resistance (AMR)-associated genes, qnrVC, sxt, bla-NDM, and bla-OXA-48 were not detected by PCR. However, the strain carried the macrolide resistance gene mphA despite showing susceptibility to the macrolide antibiotics azithromycin and erythromycin. Compared to the isolates from diarrheal patients, it was noted that although these V. cholerae strains share a similar AMR profile with SNVC-01, the strains differ significantly in other phenotypic and genetic features (Table 2).

Fig. 3

Disc diffusion assay showing susceptibility of the strain against twelve different drugs, and resistant against colistin and vancomycin

Discussion

Presently available data on sepsis incidences in Bangladesh suggests that these cases are predominantly attributed to gram-negative bacteria [27,28,29]. However, none of the studies detected V. cholerae, even though there have been recorded cases of bacteremia and septicemia caused by V. cholerae in several regions worldwide [6]. In a recent study on the ICU patients treated in icddr, b Dhaka Hospital, Sarmin et al. reported that 27% of the isolates from the stool of diarrheal patients diagnosed with severe sepsis were Vibrio spp [28]. The majority of the previous V. cholerae bacteremia case studies reported that the source of infection was either through the consumption of raw or undercooked seafood or by coming into direct contact with contaminated water while having an open wound [3, 6, 12, 13, 30,31,32]. While the exact causal relationship between enteric infections and sepsis is not completely understood, it has been shown that enteric pathogens can damage the gut mucosal layers and exacerbate intestinal infection by impacting the extraintestinal organs. Some enteric infections trigger a cascade of cellular events, ultimately causing cell death, increased gut permeability, and subsequent release of components from the host’s intestinal lumen into the circulatory system and other organs. This leads to detrimental consequences such as sepsis. Whether or not the infection has spread beyond the gastrointestinal tract, bacterial endotoxins are known to enhance the severity of sepsis, most likely by amplifying the inflammatory processes [33]. A similar observation came up in several other studies, where translocated bacteria from the inflamed gut was thought to have resulted in severe sepsis [27].

Our understanding of NOVC strains linked to human diseases is still limited. With a few exceptions, most research on bacteremia and sepsis caused by V. cholerae primarily consists of case reports and lacks a thorough analysis of the causative strains. In this study, we performed extensive genetic screening of the potential virulence genes following the identification of the associated V. cholerae strain. While the strain lacked the cholera-toxin-encoding gene ctx, toxin coregulated pili (tcpA) and porin protein-encoding gene ompU, it carried virulence genes such as type three secretion system (TTSS), repeat in toxin (RTX) gene clusters, and the gene encoding hemolysin (hlyA). TTSS has been found to cause diarrheal symptoms comparable to full-blown cholera and, hence, can serve as a potent virulence factor for NOVC [34]. TTSS could be crucial for the clinical manifestation of the study isolate. Studies on animal models show that TTSS gene clusters play a critical role in the colonization of host epithelial cells, aid in initiating an infection, and are involved in the increased pathogenic potential of V. cholerae [35, 36]. While epidemic strains of V. cholerae use TCP as the primary colonization factor, NOVC strains that do not encode TCP instead employ TTSS. In addition to TTSS, V. cholerae may also rely on ompU as an adherence factor and is recognized as an important virulence factor for its contribution to the colonization of host epithelial cells [37, 38]. We observed that the strain from the sepsis patient possesses essential translocation and effector protein-encoding genes of TTSS but does not have ompU. This indicates that TTSS may have facilitated gut colonization and led to diarrhea in the patient. RTX gene clusters have also been shown to be associated with cytotoxicity in mammalian cells [39]. Hemolysis of erythrocytes is a virulence trait widely distributed among pathogenic Vibrio spp. When tested for this property, SNVC-01 demonstrated moderate beta-hemolytic activity against sheep erythrocytes and was found to carry the V. cholerae El Tor hemolysin gene, hlyA ET. hlyA is strongly associated with invading pathogens and is involved in enhanced cytotoxicity and apoptosis during infection [11, 40]. This property of the strain would be important for future references, considering the potential association of thalassemia and hemolytic behaviour of the pathogen. Upon comparing the NOVC isolates from diarrheal patients, we found that irrespective of the phenotypic features, all the strains carried hlyA, while their hemolytic behaviour varied from no to strong hemolysis (Table 2). Other potential virulence factors might have contributed to the clinical outcome, and whole genome sequencing of the isolate will provide clues to mend the gaps in the understanding of the case. Recently, vibriobactin utilization protein subunit B (viuB) was shown to be a promising molecular marker to study the population structure and close relatives of V. cholerae as each allele of viuB roughly corresponds to a distinct lineage of V. cholerae [41]. When we compared the viuB sequence of SNVC-01 with the NCBI database, three isolates that were isolated from environmental and clinical sources shared 100% sequence identity. Considering the known diversity of the viuB genotype [19, 41] viuB-56 appears uncommon and found for the first time in Bangladesh.

Recent studies suggest that antimicrobial resistance (AMR) properties are not only associated with the disease’s clinical outcome but also potentially serve as important evolutionary markers that possibly impact the survival and adaptability of the bacteria in environments like the human body [42]. Even though the isolate SNVC-01 was found sensitive to the commonly used antibiotics in preliminary screening, considering the gravity of the case, the context of the global rise of AMR and lack of information on the epidemiological patterns of NOVC, we expanded the phenotypic and molecular tests pertinent to AMR properties additionally including some non-conventional antibiotics for V. cholerae, like vancomycin and colistin as done in several studies [43,44,45]. When the AMR profile of the isolate was compared to that of contemporary NOVC clinical isolates in our collection, few strains (CD5, CM24, CM25) isolated from diarrheal patients showed similar AMR (Table-2) pattern. Resistance to antimicrobials is a challenge for the clinical management of hospital-associated infections, including septicemia. The NOVC isolate SNVC-01 was susceptible to all the drugs that are commonly prescribed for vibriosis or bacteremia, in contrast to the contemporary multi-drug resistant V. cholerae strains prevalent in Bangladesh (Table-2). The AMR gene profile of this strain was also strikingly different from the contemporary NOVC isolates associated with clinical cases in Bangladesh. Only mphA, encoding for macrolide resistance, was detected in the strain by PCR. Despite the strain’s susceptibility to macrolide antibiotics, this gene indicates a potential functional role. Quinolone resistance in V. cholerae has been linked to mutations in the DNA gyrase gene gyrA and the topoisomerase gene parC. Over the years, the El Tor variants of V. cholerae acquired several mutations in these genes, thus serving as a marker to predict the strain type. It has recently been shown that mutations in the gyrA gene appear to be distinct among the lineages and could be an indicator of the current circulating lineage [26]. PCR amplicon sequencing of gyrA and parC revealed that SNVC-01 harbors the wild type genes and does not possess any mutations, in contrast to the contemporary O1 strains in Bangladesh [26].

Although the majority of infections caused by bacterial pathogens are treatable with available antibiotics [4] and the proportion of patients achieving complete recovery is significant, there are unfortunate incidences leading to mortality, many of which are associated with patients having pre-conditions like compromised immune systems and chronic illnesses [3, 10, 11, 13, 14] (J. Chen et al., 2020; Ding et al., 2022; Engel et al., 2016; Hwang et al., 2021; Phetsouvanh et al., 2008). Clinical manifestations of bacteremia and sepsis caused by V. cholerae can vary depending on the route of infection. The most common clinical presentations are diarrhea, abdominal discomfort, fever, and vomiting [6, 14, 30, 46]. Similarly, in the present case, the patient was suffering from abdominal pain, diarrhea, nausea, and fever. While the causal link between the severity of the disease and the pathogenic traits of the isolated bacteria is often obscure, it is widely acknowledged that host susceptibility is likely to play a significant role. Children, elderly people, individuals with liver diseases, and compromised immune systems are considered to be at higher risk of acquiring an infection with V. cholerae [14, 46]. In this context, knowing the comorbidity and plausible correlation with the disease progression is instrumental in better understanding a case. Eventhough, the patient in the present case was in rather stable condition before the infection, he had preconditions of hepatosplenomegaly and beta-thalassemia. Thalassemia is considered a risk factor for infection due to iron overload and immune abnormalities among these individuals [47]. There are several records of V. cholerae bacteremia or sepsis in individuals with thalassemia and liver disorders; nonetheless, these patients exhibited satisfactory recovery following treatment [10, 12, 30]. These cases indicate that thalassemia may not be the only causal factor for the increased probability of mortality. However, the presence of such co-morbidity can increase the host’s vulnerability; hence, selecting a treatment regimen should be approached cautiously in such cases.

There remain substantial challenges towards understanding septic associated deaths as simultaneous derangement of multiple pathways likely mediate mortality rather than a single mediator [48]. The patient in this case displayed splenomegaly which can contribute to pancytopenia and predispose the patient to septicemia [49]. Additionally, pre-existing liver dysfunction can be a contributor to the progression of infection to sepsis, multiple organ dysfunction and sepsis-induced death [50]. There are reports on hemochromatosis (iron overload) inducing gut leakage, systematic inflammation and sepsis in patients with thalassemia [51]. Adding to the confounding factors, the potential consequences of drug toxicity, which can significantly affect a patient’s condition, especially in a severely diseased state when drug metabolism could be hindered cannot be ruled out. Considering the patient’s medical history of being diagnosed with hepatosplenomegaly, it is plausible that the patient could have experienced impaired drug metabolism. However, it is challenging to attribute the adverse outcome of a patient to antibiotics since multiple factors operate in unison within the human body. For example, it is shown that carbapenems, which rely heavily on renal routes of elimination, may have a negative impact on renal function. In adverse cases, they were also associated with neutropenia and leucopenia [52]. Given the high occurrence of acute renal failure and associated death in ICU patients [53] and the possible side effects of such drugs, it is advisable to evaluate all relevant factors including pre-existing comorbidities, local drug resistance trend, ecology of the potential pathogen associated while developing policies for empirical drug usage in such cases. Hence, it is imperative to consider the potential of antibiotic-induced toxicity in sepsis, necessitating extensive research in the field.

Concluding remarks

V. cholerae has gained significance as an emerging etiology of septicemia worldwide. Yet ours was the first case of V. cholerae septicemia from Bangladesh, where cholera is endemic. The study’s major limitation may be the inability to compare and analyse the case due to a lack of data, and the measurements were made only on the day of his admission at the hospital. Nevertheless, this report bears importance as a reference case for forming comprehensive studies in the future on the burden, source, clinical, microbiological and immunological aspects of bacteremia and sepsis in Bangladesh. This would require a nationwide surveillance program involving hospitals of different categories that are representative of the burden. The detailed collection of metadata, and timely microbiological and molecular analysis should be included in the policy for managing such cases. Furthermore, there is a pressing need to implement a data-driven antimicrobial usage policy rather than empirical usage to give the best chance to our current clinical settings to save lives.